1. Field of the Invention
The present invention provides maleiimide compounds and pharmaceutical compositions comprising same. The present invention further provides methods of regulating protein phosphorylation and dephosphorylation by phosphatases inhibition and methods for treating patients suffering from cancer.
2. Background
Protein phosphorylation and dephosphorylation are important in mammalian cells for intracellular control of protein function and signaling. Two general types of mammalian protein phosphatases, enzymes that remove phosphate residues from proteins, have been identified: S/T specific (protein serine/threonine, PSTP) and Y specific (protein tyrosine, PTP). There is however an important class of dual specificity (DSP) phosphatases that is unique in its ability to dephosphorylate both phospho-tyrosine and phospho-threonine/serine on the same protein substrate (Wera, S, and B. A. Hemmings, Serine/threonine protein phosphatases. Biochem J, 1995. 311 (Pt 1): p. 17-29). The DSPs all exhibit the conserved HCX5R motif also seen in the active site of PTPs, where H is a highly conserved histidine, C is the catalytic cysteine, the five X residues form a loop in which all the amide nitrogens hydrogen-bond to the phosphate of the substrate, and R is a highly conserved arginine that hydrogen-bonds to the phosphorylated amino acid of the substrate. DSPs display a marked preference for cyclin-dependent kinases and MAP-kinases and are recognized as an important group of regulators of cell cycle control and mitogenic signal transduction. See, for example, Keyse, S. M, An emerging family of dual specificity MAP kinase phosphatases. Biochim Biophys Acta, 1995. 1265(2-3): p. 152-60; Denu, J. M., et al., Visualization of intermediate and transition-state structures in protein-tyrosine phosphatase catalysis. Proc Natl Acad Sci USA, 1996. 93(6): p. 2493-8; Galaktionov, K., X. Chen, and D. Beach, Cdc25 cell-cycle phosphatase as a target of c-myc. Nature, 1996. 382(6591): p. 511-7; and Maehama, T. and J. E. Dixon, The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem, 1998. 273(22): p. 13375-8. The Cdc25 protein phosphatases are important members of the DSPs that control cell cycle progression by activating cyclin-dependent kinases (cdk) and participate in Raf-1 mediated cell signaling (Nilsson, I. and I. Hoffmann, Cell cycle regulation by the Cdc25 phosphatase family. Prog Cell Cycle Res, 2000. 4: p. 107-14; and Xia, K., et al., Tyrosine phosphorylation of the proto-oncoprotein Raf-1 is regulated by Raf-1 itself and the phosphatase Cdc25A. Mol Cell Biol, 1999. 19(7): p. 4819-24). Three homologs of Cdc25 have been found in mammals, Cdc25A, Cdc25B and Cdc25C (Sadhu, K., et al., Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G2. Proc Natl Acad Sci USA, 1990. 87(13): p. 5139-43; Millar, J. B., et al., p55CDC25 is a nuclear protein required for the initiation of mitosis in human cells. Proc Natl Acad Sci USA, 1991. 88(23): p. 10500-4; and Nagata, A., et al., An additional homolog of the fission yeast cdc25+ gene occurs in humans and is highly expressed in some cancer cells. New Biol, 1991. 3(10): p. 959-68). Cdc25A is important for G1/S phase transition (Jinno, S., et al., Cdc25A is a novel phosphatase functioning early in the cell cycle. Embo J, 1994. 13(7): p. 1549-56), although it may also have some role in the initiation of mitosis (Molinari, M., et al., Human Cdc25A inactivation in response to S phase inhibition and its role in preventing premature mitosis. EMBO Rep, 2000. 1(1): p. 71-9). Both Cdc25B and Cdc25C are regulators of G2-M transition and S-phase progression (Lammers, R., et al., Differential activities of protein tyrosine phosphatases in intact cells. J Biol Chem, 1993. 268(30): p. 22456-62). Cdc25A and Cdc25B are over expressed in many cancers and are associated with poor prognosis (Galaktionov, K., X. Chen, and D. Beach, Cdc25 cell-cycle phosphatase as a target of c-myc. Nature, 1996. 382(6591): p. 511-7; Galaktionov, K, et al., CDC25 phosphatases as potential human oncogenes. Science, 1995. 269(5230): p. 1575-7; Gasparotto, D., et al., Overexpression of CDC25A and CDC25B in head and neck cancers. Cancer Res, 1997. 57(12): p. 2366-8; Hernandez, S., et al., cdc25 cell cycle-activating phosphatases and c-myc expression in human non-Hodgkin's lymphomas. Cancer Res, 1998. 58(8): p. 1762-7; and Wu, W., et al., Overexpression of cdc25A and cdc25B is frequent in primary non-small cell lung cancer but is not associated with overexpression of c-myc. Cancer Res, 1998. 58(18): p. 4082-5). Potent and selective inhibitors of Cdc25 would thus be attractive candidates as potential anticancer agents.
A few Cdc25 phosphatase inhibitors have been reported to date: dephostatin, sulfurcin, dnacin A1 and B1, vitamin K3 and analogs and other naphthoquinone analogs, azido-homo-oxa steroid, alkyllysophospholipid analogs, and coscinosulfate. See, for example, Imoto, M, et al., Dephostatin, a novel protein tyrosine phosphatase inhibitor produced by Streptomyces. I. Taxonomy, isolation, and characterization. J Antibiot (Tokyo), 1993. 46(9): p. 1342-6; Cebula, R. E. B., J. L.; Boisclair, M. D.; Mansuri, M. M.; Pal, K.; Bockovich, N. J., Synthesis and phosphatase inhibitory activity of analogs of sulfurcin. Bioorg. Med. Chem. Lett, 1997. 7: p. 2015-2020; Horiguchi, T, et al., Dnacin A1 and dnacin B1 are antitumor antibiotics that inhibit cdc25B phosphatase activity. Biochem Pharmacol, 1994. 48(11): p. 2139-41; Borgne, A. and L. Meijer, Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition. J Biol Chem, 1996. 271(44): p. 27847-54; Tamura, K., et al., Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue. Cancer Res, 2000. 60(5): p. 1317-25; Ham, S. W., et al., Naphthoquinone analogs as inactivators of cdc25 phosphatase. Bioorg Med Chem Lett, 1998.8(18): p. 2507-10; Peng, H., et al., Novel CDC25A phosphatase inhibitors from pyrolysis of 3-alpha-azido-B-homo-6-oxa-4-cholesten-7-one on silica gel. J Med Chem, 1998. 41(24): p. 4677-80; Koufaki, M., et al., Alkyl and alkoxyethyl antineoplastic phospholipids. J Med Chem, 1996. 39(13): p. 2609-14; Loukaci, A. S., Isabelle Le; Samadi, Mohammad; Leclerc, Sophie; Damiens, Eve; Laurent, Meijer; Debitus, Cecile; Guyot, Michele, Coscinosulfate, a CDC25 Phosphotase Inhibitor from the Sponge Coscinoderma Mathewsi. Bioorg. Med. Chem. Lett, 2001. 9: p. 3049-3054.
An attractive feature of vitamin K3 and its analogs as a broad spectrum antitumor agents lie with their relatively low toxicity (LC50 for Vitamin K3 orally in mice, 0.5 g/kg) in comparison with other quinone antitumor agents. The synthetic vitamin K3 analogs, are more potent growth inhibitors in human Hep3B cell line and inhibitors of DNA synthesis in normal rat hepatocytes. However, the activity was generally in the low micromolar range. We synthesized several new vitamin K analogs, which were only marginally better than the best non-toxic compound that was available Cpd 5,2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone.

Improvements in the activity of vitamin K analogs as phosphatase inhibitors is expected to come at the expense of greater general toxicity since the vitamin K compounds are quinines, which are known to be generally toxic because of redox cycling and the concomitant formation of reduced oxygen species.
Thus, it would be desirable to provide new vitamin K analogs which do not contain a naphthoquinone ring system and are thus avoid the toxicity associated with vitamin K naphthaquinone compounds due to redox cycling. Moreover, it would be desirable to provide new compounds having high affinity and selectivity for a variety of phosphatase enzymes, or more particularly DSP enzymes. It would be further desirable to provide a new class of phosphatase inhibitors which are easy to prepare in a minimal number of steps. It would also be desirable to have methods of inhibiting phosphatase and methods of treating a variety of cancers using the new non-naphthaquinone vitamin K analogs.